Solid phase extraction (SPE) is a chromatographic technique for preparing samples prior to performing quantitative chemical analysis, for example, via high performance liquid chromatography (HPLC), or gas chromatography (GC). The goal of SPE is to isolate target analytes from a complex sample matrix containing unwanted interferences, which would have a negative effect on the ability to perform quantitative analysis. The isolated target analytes are recovered in a solution that is compatible with quantitative analysis. This final solution containing the target compound can be directly used for analysis or evaporated and reconstituted in another solution of a lesser volume for the purpose of further concentrating the target compound, making it more amenable to detection and measurement.
Depending on the type of analysis to be performed, and detection method used, SPE may be tailored to remove specific interferences. Analysis of biological samples such as plasma and urine using high performance liquid chromatography (HPLC) generally requires SPE prior to analysis both to remove insoluble matter and soluble interferences, and also to pre-concentrate target compounds for enhanced detection sensitivity. Many sample matrices encountered in bio-separations contain buffers, salts, or surfactants, which can be particularly troublesome when mass spectrometer based detection is used. SPE can also be used to perform a simple fractionation of a sample based on differences in the chemical structure of the component parts, thereby reducing the complexity of the sample to be analyzed.
Devices designed for SPE typically include a chromatographic sorbent which allows the user to preferentially retain sample components. Once a sample is loaded onto the sorbent, a series of tailored washing and elution fluids are passed through the device to separate interferences from target sample components, and then to collect the target sample components for further analysis. SPE devices usually include a sample holding reservoir, a means for containing the sorbent, and a fluid conduit, or spout for directing the fluids exiting the device into suitable collection containers. The SPE device may be in a single well format, which is convenient and cost effective for preparing a small number of samples, or a multi-well format, which is well suited for preparing large numbers of samples in parallel. Multi-well formats are commonly used with robotic fluid dispensing systems. Typical multi-well formats include 48-, 96-, and 384-well standard plate formats. Fluids are usually forced through the SPE device and into the collection containers, either by drawing a vacuum across the device with a specially designed vacuum manifold, or by using centrifugal or gravitational force. Centrifugal force is generated by placing the SPE device, together with a suitable collection tray, into a centrifuge specifically designed for the intended purpose.
Various means have been used to contain chromatographic sorbents within SPE devices. A common method, described in U.S. Pat. No. 4,211,658, utilizes two porous filters, with chromatographic sorbent contained between the filters. In this design, the SPE device is essentially a small chromatographic column containing a packed bed of sorbent. A variation of this design is described in U.S. Pat. No. 5,395,521, where the porous filter elements are spherical in shape. In U.S. Pat. No. 4,810,381, the chromatographic sorbent is immobilized within a thin porous membrane structure. In EP Application No. 1110610 A1 a method is described for securing these filters within the SPE device by means of a sealing ring pressed around the periphery of the membrane disc. In U.S. Pat. No. 5,486,410 a fibrous structure containing immobilized functional materials is described. In U.S. Pat. No. 5,906,796 an extraction plate is described where glass fiber discs containing chromatographic sorbent are press fit into each well of the SPE device.
A number of chromatographic sorbents can be used depending on the nature of the sample matrix and target compounds. A common example is to use porous silica that has been surface derivatized with octydecyl (C18) or octyl (C8) functional groups. Porous particles that are based on organic polymers are also widely used. One such type, which is particularly well suited for SPE due to its high loading capacity and unique retention properties, is described in U.S. Pat. No. 6,254,780.
Typical SPE methods contain a sequence of steps, each with a specific purpose. The first step, referred to as the xe2x80x9cconditioningxe2x80x9d step, prepares the device for receiving the sample. For reversed-phase SPE, the conditioning step involves first flushing the SPE device with an organic solvent such as methanol or acetonitrile, which acts to wet the surfaces of both the device and the sorbent, and also rinses any residual contaminants from the device. This initial rinse is generally followed with a highly aqueous solvent rinse, often containing pH buffers or other modifiers, which will prepare the chromatographic sorbent to preferentially retain the target sample components. Once conditioned, the SPE device is ready to receive the sample.
The second step, referred to as the xe2x80x9cloadingxe2x80x9d step, involves passing the sample through the device. During loading, the sample components, along with many interferences are adsorbed onto the chromatographic sorbent. Once loading is complete, a xe2x80x9cwashingxe2x80x9d step is used to rinse away interfering sample components, while allowing the target compounds to remain retained on the sorbent. The washing step is then followed by an xe2x80x9celutionxe2x80x9d step, which typically uses a fluid containing a high percentage of an organic solvent, such as methanol or acetonitrile. The elution solvent is chosen to effectively release the target compounds from the chromatographic sorbent, and into a suitable sample container.
In many cases, elution with high concentrations of organic solvent requires that further steps be taken before analysis. In the case of chromatographic analysis (HPLC), it is highly desirable for samples to be dissolved in an aqueous-organic mixture rather than a pure organic solvent, such as methanol or acetonitrile. For this reason, SPE samples eluted in pure acetonitrile or methanol are usually evaporated to dryness (xe2x80x9cdrydownxe2x80x9d), and then reconstituted in a more aqueous mixture (xe2x80x9creconstitutionxe2x80x9d) before being injected into an HPLC system. These additional steps not only take time and effort, but can also lead to loss of valuable sample, either through target analyte loss onto collection container surfaces during drydown, or due to target analyte evaporative losses or difficulties encountered when trying to re-dissolve the dried sample in the higher percent aqueous fluid.
It can be seen then, that it is advantageous for an SPE device to have a high capacity for retaining target compounds of a wide range of chromatographic polarities, to be capable of maintaining target compound retention as sample interferences are washed to waste, and then to provide the capability to elute target compounds in as small an elution volume as possible, thereby maximizing the degree of target compound concentration obtained during SPE.
The ability to elute in very small volumes of solvent has the added benefit of minimizing the amount of time required to evaporate and reconstitute the sample before proceeding with analysis if further concentration or solvent exchange is required. If elution volume can be kept very low, then drydown and reconstitution can be entirely eliminated.
Traditional SPE device designs have attempted to address these issues, each with a limited measure of success. Packed bed devices utilize packed beds of sorbent particles contained between porous filter discs that are press fit into the SPE device. The capture efficiency of the resulting packed beds is typically quite good, especially if the sorbent properties are carefully chosen. One drawback with conventional packed bed devices is that the void volume contained within the porous filters and packed bed requires that relatively large elution volumes be used to completely elute the target compounds. Typical elution volumes required to fully elute target compounds from a packed bed type SPE device fall in the range of 200-5000 xcexcL, depending on the size of the sorbent bed.
Membrane based designs attempt to address this issue by embedding sorbent particles within a fluorocarbon based membrane, which are then placed into the SPE device. A small mass of sorbent particles is embedded into a thin membrane structure with a wide cross sectional area. Since the membrane does not require retaining filters, the volume associated with the two porous filters is eliminated. This approach reduces the total volume contained within the device, and therefore the volume of solvent required for elution. A typical elution volume required to fully elute target compounds from a particle in membrane SPE device fall in the range of 75-500 xcexcL. Designs of this type have drawbacks in other areas however. The sorbent particles are less densely packed within the membrane structure than within a packed bed, leading to poorer capture efficiency, and a greater chance that target compounds will break through the device without being well retained. In addition, the flow properties of the membrane can be highly variable, due to the poor wetting characteristics of the fluorocarbon based membrane when using highly aqueous fluids.
In U.S. Pat. No. 5,906,796 a design is described in which glass fiber based extraction discs containing chromatographic particles are press fit into each well of the SPE device. Like the membrane designs, this approach immobilizes the sorbent particles in a thin sheet, thereby minimizing device void volume and required elution volumes. Typical volumes required to fully elute target compounds from an SPE device such as this fall in the range of 75-500 xcexcL, which is comparable to particle-in-membrane devices. The sorbent particles are even less densely packed than with membranes however, so sample breakthrough tends to be higher than with either membrane or packed bed devices, and sorbent particles can often break free from the fibrous matrix and contaminate the collected sample solution. One advantage over membrane devices is that flow problems due to wetting issues are generally less common due to the more open structure of the glass fiber disc. One disadvantage of this particle embedded glass fiber disk is that it contains silanol groups that interact with basic compounds. This requires the use of more complex elution solvents, for example, the addition of 2% base or acid to the elution solvent, to maintain the 75-500 xcexcL elution volumes.
It can be seen then, that the lower elution volume capability achieved with both the membrane and glass fiber approaches is at the expense of target compound breakthrough during loading and/or poor recovery for non-polar compounds. Although the volume of fluid needed to effectively elute samples from the membrane and glass fiber formats is reduced to approximately one half of the volume required with conventional packed bed based devices, dry-down and reconstitution steps are still required before samples can be further analyzed by HPLC.
The present invention relates to an improved SPE device which has been specifically designed to contain a small packed bed of chromatographic sorbent such that the bed provides for highly efficient retention of target compounds, while the volume contained within the sorbent bed is sufficiently small as to allow for efficient elution of sample compounds in a minimal elution volume. Specifically, the solid phase extraction device of the present invention comprises a reservoir with an opening; a well comprising an internally tapered wall, the well having a wider interior diameter at an end closest to the opening than at an exit spout; a first filter within the well; a bed of sorbent particles within the well below the first filter; and a second filter having a smaller diameter than the first filter within the well below the bed of sorbent particles and above the exit spout.
The present invention provides a large bed height to top bed diameter ratio using a significantly smaller sorbent mass than is present in current state of the art devices. The large bed height to bed diameter ratio enhances the retention of target compounds and helps to prevent breakthrough of these compounds during the load and wash steps. In SPE the first filter and the top of the sorbent bed acts like a depth filter in removing insoluble sample components. The larger diameter for the upper portion of the bed and larger diameter first filter allows the device to draw through larger sample volumes than could be drawn through a device having an upper bed diameter the same as the lower bed diameter before obstructions will occur. The smaller second filter increases the bed height to bed diameter ratio for a given mass of sorbent while reducing the hold up volume of the device which minimizes required elution volumes.
Moreover, the present invention provides for conically shaped packed beds contained between spherical filters which enhance the performance of solid phase extraction devices by allowing target compounds to be both efficiently retained and eluted. The larger first spherical filter provides a surface area that is approximately two times the area of an equivalently sized cylindrical filter. For example, surface area of the top half of a sphere (xcfx80/2xc3x97d2) of a diameter of 0.1xe2x80x3 is equal to the surface area of the top of a disk of diameter 0.14xe2x80x3 (xcfx80/4xc3x97d2). The smaller second filter helps to minimize the amount of sorbent needed to create a bed length that will be free of adverse imperfections.
The present invention enables the retention of target compounds with a wide range of chromatographic polarity with elution in volumes that are much reduced from the current state of the art for solid phase extraction. This reduction in elution volume provides a solution containing the target compounds that can be diluted with an aqueous solution while still maintaining the high sample concentrations required for analysis.
According to another aspect, the present invention provides an enhanced method of performing solid phase extraction, where the volume of elution solvent is sufficiently small so as to eliminate the need for an evaporation step. The method involves elution of the target compounds in a minimal volume of organic solvent, typically 10-40 xcexcL, which is then diluted with a highly aqueous fluid to form an aqueous organic sample mixture. This mixture is suitable for direct analysis by HPLC, thereby eliminating the time, expense, and potential sample losses associated with evaporation and reconstitution steps, while still maintaining a high degree of target compound(s) concentration.
Specifically, the inventive method comprises the steps of providing the above-mentioned SPE device, and isolating target substances from interfering components in a sample medium, wherein the target substances are substantially eluted in less than 50 xcexcL volume.
In one embodiment of the present invention, the isolating step of the present invention preferably includes conditioning the SPE device with an organic solvent; equilibrating the SPE device with an aqueous solution; adding a prepared sample containing the target substances and interfering components to the SPE device; washing the SPE device with an aqueous solution to remove interfering components; and eluting the adsorbed target substances.
In a preferred embodiment of this enhanced method of the present invention, the aqueous diluent is added directly through the SPE device, while still on the processing station used to perform the SPE fluid transfers. In this way, residual elution solvent is swept through the device into the collection container, where it is diluted by the aqueous fluid and mixed by the gentle air stream that is drawn through the well at the end of the transfer. This approach has the advantage of eliminating the need for a separate pipetting operation to perform the dilution step.